Insulin Chemiluminescence ELISA For the quantitative determination of insulin in human Serum, Heparin Plasma, and Tissue Culture Supernatants. For In Vitro Diagnostic use within the United States of America. This product is for Research Use Only outside of the United States of America. Catalog Number: Size: 80-INSHU-CH01, CH10 96 Wells Version: May 29, 2018 26-G Keewaydin Drive, Salem, NH 03079 P: (800) 592-5726 F: (603) 898-6854 ts@alpco.com www.alpco.com
INTEED USE The ALPCO Insulin Chemiluminescence ELISA is designed for the quantitative determination of insulin in human serum, heparin plasma, and tissue culture supernatants. PRINCIPLE OF THE ASSAY The ALPCO Insulin Chemiluminescence ELISA is a sandwich type immunoassay. The 96-well microplate is coated with a monoclonal antibody specific for insulin. The standards, controls, and samples are added to the microplate wells with the conjugate. The microplate is then incubated at room temperature on a microplate shaker at 700-900 rpm. After the first incubation is complete, the wells are washed with Wash Buffer and blotted dry. SA-HRP is added to the wells for a second incubation at room temperature on a microplate shaker at 700-900 rpm. The wells are washed with Wash Buffer and blotted dry. Chemiluminescent Substrate is added, and the microplate is read by a luminescence plate reader after 5 minutes. The intensity of the light generated is directly proportional to the amount of insulin in the sample. MATERIALS SUPPLIED 80-INSHU-CH01 Component Quantity Preparation Insulin Microplate (96 wells) 12 x 8 strips Ready to use Zero Standard 5 ml Ready to use Standards (A-G) (30,000, 10,000, 1,000, 100, 20, 10, 5 pg/ml) (862.1, 287.4, 28.74, 2.874, 0.575, 0.287, 0.144 µiu/ml) 1 ml each Ready to use Control Levels 1, 2 and 3* 1 vial each Lyophilized* Conjugate 0.15 ml 101X Conjugate Buffer 9 ml Ready to use SA-HRP 0.15 ml 101X SA-HRP Buffer 12 ml Ready to use Wash Buffer Concentrate 100 ml 21X Substrate A 6 ml Ready to use Substrate B 6 ml Ready to use Plate Sealers 3 Ready to use Insulin Chemiluminescence ELISA Page 2 of 13 May 29, 2018
80-INSHU-CH10 Component Quantity Preparation Insulin Microplate (96 wells) 10X (12 x 8 strips) Ready to use Zero Standard 5 ml Ready to use Standards (A-G) (30,000, 10,000, 1,000, 100, 20, 10, 5 pg/ml) (862.1, 287.4, 28.74, 2.874, 0.575, 0.287, 0.144 µiu/ml) 1 ml each Ready to use Control Levels 1, 2 and 3* 1 vial each Lyophilized* Conjugate 1 ml 101X Conjugate Buffer 90 ml Ready to use SA-HRP 1.5 ml 101X SA-HRP Buffer 120 ml Ready to use Wash Buffer Concentrate 2 x 200 ml 21X Substrate A 60 ml Ready to use Substrate B 60 ml Ready to use Plate Sealers 20 Ready to use *Please refer to the Certificate of Analysis enclosed with each kit for more information. MATERIALS REQUIRED Precision pipettes for dispensing 10-100 µl (with disposable tips) Repeating or multi-channel pipette for dispensing up to 100 µl Volumetric containers and pipettes for reagent preparation Distilled or deionized water for reagent preparation Microplate washer or wash bottle Microplate shaker capable of 700-900 rpm Microplate reader with luminometer PRECAUTIONS 1. The human blood products incorporated into this kit have been tested for the presence of HIV (Human Immunodeficiency virus), HBV (Hepatitis B virus), and HCV (Hepatitis C virus). Test methods for these viruses do not guarantee the absence of a virus; therefore, all reagents should be treated as potentially infectious. Handling and disposal should be in accordance with all appropriate national and local regulations for the handling of potentially biohazardous materials. 2. All materials derived from animal sources are BSE negative. However, all materials should be treated as potentially infectious. 3. Avoid direct contact with skin. 4. This product is not for internal use. 5. Avoid eating, drinking, or smoking when using this product. Insulin Chemiluminescence ELISA Page 3 of 13 May 29, 2018
6. Do not pipette any reagents by mouth. 7. Reagents from this kit are lot-specific and must not be substituted. 8. Do not use reagents beyond the expiration date. 9. Variations to the test procedure are not recommended and may influence the test results. 10. An appendix has been included with examples of instrument settings for reading a chemiluminescent output. Each lab should optimize their instrument settings according to the manufacturer s instructions. Please contact the technical services department of the manufacturer of the microplate reader for optimal instrument settings. STORAGE COITIONS The kit should be stored at 2-8 C. The kit is stable until the expiration date on the box label. SAMPLE HALING Serum and plasma samples are appropriate for use in this assay. No dilution or treatment of the sample is required. However, if a sample has a greater concentration of insulin than the highest standard, the sample should be diluted in Zero Standard and the analysis should be repeated. Tissue culture supernatants and serum-free tissue culture media are also appropriate for use in this assay. Tissue culture media containing serum (complete media) should be diluted 1:16 for use in this assay. It is recommended to 1) thoroughly vortex each sample before use, 2) spin the samples before use, and 3) perform pipetting actions without pausing. Samples can be stored at 2-8 C for 24 hours prior to analysis in this assay. For longer periods, storage at < -20 C is recommended. Avoid repeated freezing/thawing of the sample REAGENT PREPARATION All reagents must be equilibrated to room temperature prior to preparation and subsequent use in the assay. Prepare reagents according to the number of plates or strips to be used. Store the remaining concentrates at 2-8ºC. Controls (Levels 1, 2 and 3) are provided in a lyophilized form. Please refer to the Certificate of Analysis provided with each kit for the appropriate volume of deionized water for reconstitution. Close the vial with the rubber stopper and cap, gently swirl the vial, and allow it to stand for 30 minutes prior to use. The contents of the vial should be in solution with no visible particulates. The reconstituted controls can be stored at -20 C in aliquots for up to 6 months. The controls should not be repeatedly frozen and thawed. Insulin Chemiluminescence ELISA Page 4 of 13 May 29, 2018
Conjugate Stock (101X) is to be diluted with 100 parts Conjugate Buffer. Working Strength Conjugate is stable for 8 hours at 2-8 C. Number of plates Amount of Concentrate Amount of Conjugate Buffer 0.5 (6 strips) 45 µl 4.5 ml 1 (12 strips) 90 µl 9.0 ml 2 180 µl 18 ml 5 450 µl 45 ml 10 900 µl 90 ml SA-HRP Concentrate (101X) is to be diluted with SA-HRP Buffer. Working Strength SA-HRP is stable for 8 hours at 2-8 C. Number of plates Amount of Concentrate Amount of SA-HRP Buffer 0.5 (6 strips) 60 µl 6 ml 1 (12 strips) 120 µl 12 ml 2 240 µl 24 ml 5 600 µl 60 ml 10 1200 µl 120 ml Wash Buffer Concentrate (21X) is to be diluted with 20 parts distilled water. Working Strength Wash Buffer is stable for 4 weeks at room temperature. NOTE: All labs should account for plate washer void volume to prime the machine. The values below do not account for priming automated plate washers. Number of plates Amount of Concentrate Amount of dh2o (6 x 350 µl wash) (6 x 350 µl wash) 0.5 (6 strips) 15 ml 300 ml 1 (12 strips) 30 ml 600 ml 2 50 ml 1,000 ml 5 130 ml 2,600 ml 10 250 ml 5,000 ml Substrates A & B are provided individually and should be combined in equal parts to create the Working Chemiluminescent Substrate prior to use. The Working Chemiluminescent Substrate is stable for 1 hour at room temperature. Number of plates Amount of Substrate A Amount of Substrate B 0.5 (6 strips) 3 ml 3 ml 1 (12 strips) 6 ml 6 ml 2 12 ml 12 ml 5 30 ml 30 ml 10 60 ml 60 ml QUALITY CONTROL It is recommended that the Controls provided with the Insulin Chemiluminescence ELISA be included in every assay. The concentration ranges of the controls are provided on the Certificate of Analysis provided with each kit; however, it is recommended that each laboratory establishes its own acceptable ranges. Insulin Chemiluminescence ELISA Page 5 of 13 May 29, 2018
ASSAY PROCEDURE All reagents and microplate strips should be equilibrated to room temperature prior to use. Gently mix all reagents before use. A standard curve must be performed with each assay run and with each microplate if more than one is used at a time. All standards, controls, and samples should be run in duplicate. A suggested plate layout is provided. 1. The microplate should be equilibrated to room temperature prior to opening the foil pouch. Designate enough microplate strips for duplicate determinations of the standards, controls, and samples. The remaining microplate strips should be stored at 2-8 C in the tightly sealed foil pouch containing the desiccant. 2. Pipette 25 µl of each standard, control, and sample into their respective wells. See Reagent Preparation and Certificate of Analysis for control reconstitution instructions. 3. Pipette 75 µl of Working Strength Conjugate (see Reagent Preparation) into each well. 4. Cover microplate with a plate sealer and incubate for 1 hour at room temperature, shaking at 700-900 rpm on a microplate shaker. 5. Decant the contents of the wells and wash the microplate 6 times with 350 µl of Working Strength Wash Buffer per well (see Reagent Preparation) using a microplate washer. a. Alternatively, fill the wells with Working Strength Wash Buffer using a wash bottle equipped with a wash nozzle or manual washer. (It is not recommended to use a multichannel pipette. Wash buffer must be dispensed with adequate and equal force in order to properly wash the wells.) Soak the wells for 1 minute. Invert the microplate to discard the liquid and firmly tap the inverted microplate on absorbent paper towels. Repeat the wash and soak procedure 2 more times, for a total of 3 washes. After the final wash, (automated or manual), remove any residual Wash Buffer and bubbles from the wells by inverting and firmly tapping the microplate on absorbent paper towels. 6. Pipette 100 µl of Working SA-HRP Solution into each well. 7. Cover microplate with a plate sealer and incubate for 30 minutes at room temperature, shaking at 700-900 rpm on a microplate shaker. 8. Decant the contents of the wells and wash the microplate 6 times with 350 µl of Working Strength Wash Buffer per well. See Step 5. 9. Pipette 100 µl of Working Chemiluminescent Substrate (see Reagent Preparation) into each well. 10. Place the microplate in a microplate reader capable of reading the luminosity of the wells. The microplate should be analyzed at 5 minutes after the addition of the Chemiluminescent Substrate, and no more than 15 minutes after substrate addition. Read the plate using a 1 second integration time. Insulin Chemiluminescence ELISA Page 6 of 13 May 29, 2018
CALCULATION OF RESULTS Construct a standard curve from the standards. It is recommended to use a software program to calculate the standard curve and to determine the concentration of the samples. A 5-parameter curve fit with 1/y 2 weighting is recommended for data analysis. The Insulin Chemiluminescence ELISA is a ligand binding assay, with responses exhibiting a sigmoidal relationship to the analyte concentration. Currently accepted reference models for such curves use a 4 or 5-parameter logistic (pl) fit, as these models optimize the accuracy and precision across a greater range. A 5-pl curve fit with 1/y 2 weighting is used to maximize the accuracy and precision of samples with low concentrations. However, the accuracy and precision of all models are limited at the lowest and highest ends of the detectable range due to the influence of individual laboratory conditions. As a result, caution should always be used when interpreting results where the analyte response becomes non-linear. 1 Extrapolating sample concentration values outside the range of the standard concentration values is not recommended. TYPICAL STAARD CURVE The following results are provided for demonstration purposes only and cannot be used in place of data obtained with the assay. A standard curve must be performed with each assay run and plate tested. A 5-parameter curve fit with 1/y 2 weighting is used for data analysis. Standard Conc. Conc. pg/ml µiu/ml RLU A 30,000 862.1 500,748 B 10,000 287.4 284,292 C 1,000 28.74 32,954 D 100 2.874 2,703 E 20 0.575 516 F 10 0.287 281 G 5 0.144 149 Zero 0 0 63 Insulin Chemiluminescence ELISA Page 7 of 13 May 29, 2018
EXPECTED VALUES The Insulin Chemiluminescence ELISA is calibrated to the WHO insulin 1 st IRP 66/304. Conversion for Human Insulin from pg/ml to µiu/ml (micro International Units): 34.8 pg/ml = 1 µiu/ml 34.8 µg = 1 IU = 6 nmol ASSAY DEVELOPMENT TIME It is recommended to read the plate 5-13 minutes after the addition of the substrate to the wells. Concentration values have been shown to have less than 3% CV from 5-15 minutes. 5 minutes 7 minutes Concentration (pg/ml) 9 11 13 minutes minutes minutes 15 minutes Mean (pg/ml) CV (%) Sample 1 391 388 398 406 405 389 396 2.0 Sample 2 5,533 5,562 5,637 5,651 5,584 5,688 5,609 1.1 Sample 3 19,739 20,114 20,331 20,411 20,403 21,377 20,396 2.7 Insulin Chemiluminescence ELISA Page 8 of 13 May 29, 2018
PERFORMANCE CHARACTERISTICS (Refer to the lot specific Certificate of Analysis for the most relevant performance data.) Sensitivity Limit of Blank (LoB) is defined as the concentration obtained by plotting the mean RLU + 1.645*SD of 20 Zero standard replicates against the standard curve. Limit of Detection (LoD) is defined as the concentration obtained by plotting the calculated LoB RLU + 1.645*SD of the same 20 replicates. The LoB and LoD of the assay are 2.0 pg/ml Limit of Quantitation (LoQ) is defined as the lowest concentration which has a calculated concentration CV 20% and an accuracy of 80-120%. The LoQ of the assay is 20 pg/ml. Precision: Within run (intra-assay) variation The within run precision is expressed as the percentage coefficient of variation (CV (%)). This was determined based on the mean and standard deviation of 16 replicates of a sample run in a single assay. The table below shows the results of 3 samples that span the range of the assay. Sample 1 Sample 2 Sample 3 Mean 21.2 pg/ml 996 pg/ml 16,846 pg/ml CV (%) 8.7 5.3 12.7 n 16 16 16 Precision: Between run (inter-assay) variation The between run precision is expressed as the percentage coefficient of variation (CV %). This was determined based on the mean and standard deviation across 2 assays of 36 measurements of a single sample. The table below shows the results of 3 samples that span the range of the assay. Sample 1 Sample 2 Sample 3 Mean 23.2 pg/ml 991 pg/ml 16,688 pg/ml CV (%) 11.6 5.3 13.3 n 32 32 32 Linearity The linearity of the assay was determined by preparing dilutions of samples with high insulin concentrations 1:2 to 1:128 with the Zero Standard. The expected values were compared to the observed values to determine a percent recovery. The mean recovery for serum samples is 98% (range 78-128%). The mean recovery for heparin plasma samples is 99% (range 87-122%). The mean recovery of islet cell culture supernatants is 86% (range 72-106%). Insulin Chemiluminescence ELISA Page 9 of 13 May 29, 2018
Spike and Recovery The spike and recovery of the assay was determined by adding various known amounts of insulin to samples. Serum, heparin plasma, cultured media, serum-free media, and complete media at 1:16 were evaluated. The expected values were compared to the observed values to determine a percent recovery. The range of recovery for the samples: Mean recovery (%) Min (%) Max (%) Serum low 94 91 100 mid 90 88 96 high 84 77 90 Heparin plasma low 91 80 97 mid 94 78 103 high 93 79 113 Cell culture supernatant low 89 76 94 mid 90 82 96 high 90 86 96 Specificity The table below indicates the analyte and the percent cross-reactivity observed in the assay. Analyte Cross-reactivity Human Insulin 100% Porcine Insulin 260% Rat Insulin 1 0.49% Rat Insulin 2 Mouse Insulin Humulin 162% Novolin 152% Insuman Basal 240% Lantus 4% Lispro Human Proinsulin Intact Human Proinsulin Des (31,32) 0.73% Human Proinsulin Des (64,65)* 65.5% Bovine Proinsulin 0.73% Porcine Proinsulin 0.86% Rat Proinsulin 1 Rat Proinsulin 2 Mouse Proinsulin 1 Mouse Proinsulin 2 Insulin Chemiluminescence ELISA Page 10 of 13 May 29, 2018
Human C-Peptide Porcine C-Peptide Canine C-Peptide Rat C-Peptide 1 Rat C-Peptide 2 Mouse C-Peptide 1 Mouse C-Peptide 2 = not detectable *Physiologically, Des (64,65) comprises of only ~6% of circulating total proinsulin. 2, 3 Hook Effect No high dose hook effect was observed with insulin concentrations up to 100,000 pg/ml. REFERENCES 1. Finlay JWA, Dillard RF. Appropriate Calibration Curve Fitting in Ligand Binding Assays. AAPS Journal. 2007; 9(2): E260-E267. Insulin Chemiluminescence ELISA Page 11 of 13 May 29, 2018
SHORT ASSAY PROTOCOL Add 25 µl standards, controls, and samples Add 75 µl Conjugate Incubate for 1 hour at RT, shake at 700-900 rpm Wash 6 times Add 100µL SA-HRP Solution Incubate for 30 minutes at RT, shake at 700-900 rpm Wash 6 times Add 100 µl Working Chemiluminescent Substrate Read luminosity at 5 minutes Total Time = 1 hour 35 minutes Insulin Chemiluminescence ELISA Page 12 of 13 May 29, 2018
SUGGESTED PLATE LAYOUT Below is a suggested plate layout for running standards, controls, and up to 37 samples in duplicate. 1 2 3 4 5 6 7 8 9 10 11 12 A Std A Std A Ctrl 1 Ctrl 1 6 6 14 14 22 22 30 30 B Std B Std B Ctrl 2 Ctrl 2 7 7 15 15 23 23 31 31 C Std C Std C Ctrl 3 Ctrl 3 8 8 16 16 24 24 32 32 D Std D Std D 1 1 9 9 17 17 25 25 33 33 E Std E Std E 2 2 10 10 18 18 26 26 34 34 F Std F Std F 3 3 11 11 19 19 27 27 35 35 G Std G Std G 4 4 12 12 20 20 28 28 36 36 H Zero Zero 5 5 13 13 21 21 29 29 37 37 Std= Standard Ctrl = Control Numbered wells = Samples APPEIX Instrument settings: Please contact the microplate reader manufacturer s technical services department for additional assistance. These instrument settings are to be used as a guideline. It is optional to shake the plates before reading for 3 seconds. Molecular Devices Spectramax L Read Mode: Luminescence Integration Time: 1 second (1000 ms) Sensitivity: PMT: MaxRange Target Calibration Wavelength: 470 nm Automix: Classic: 30 mm/s Automix before read: Off Plate Type: 96 well standard Injection and Delay: Off Injection wells: None Dark Adapt: Off AutoRead: Off Biotek Synergy2 Detection Method: Luminescence Read Type: Endpoint Integration: 0:01.00 (MM:SS.ss) (1 second) Emission: Hole Optics Position: Top Sensitivity: 150 Top Probe Vertical Offset: 1.00mm Molecular Devices Spectramax M5 Read Mode: Luminescence (LUM) Read Type: Endpoint Wavelength: All Plate Type: 96 well standard opaque Read Area: Variable based on experiment PMT and Optics: Integration Time 1000 ms Shake: Off More Settings: Calibrate (on); Carriage Speed (Normal); Read Order (Column); Setting Time (off) Tecan Infinite 200 Plate: Corning 96 Flat Bottom Black Polystyrol Mode: Luminescence Attenuation: NONE Integration Time: 1000 ms Settle Time: 0 ms Insulin Chemiluminescence ELISA Page 13 of 13 May 29, 2018